Primary colorectal cancers (CRC) demonstrate significant variability in expression of granulocyte colony stimulating factor and receptor (GCSF/R), yet the effect of these varying levels of expression on the metastatic potential of CRC is unknown. The long-term goal of this work is to discover and test new therapeutic targets for CRC based on an improved understanding of the tumor microenvironment. The overall objective of this project is to determine the extent to which elevated GCSF signaling increases metastasis in CRC, and to interrogate the underlying causal mechanisms. The central hypothesis is that increased GCSF signaling promotes CRC metastasis. The rationale for the proposed research is that an understanding of how GCSF signaling may enhance CRC metastatic potential will lay critical groundwork for consideration of GCSF as a therapeutic target for this lethal disease. Guided by strong preliminary data, this hypothesis will be tested by completing three specific aims: (1) determine the extent to which serum GCSF levels and metastatic tumor expression of GCSF and GCSFR correlate with outcomes in CRC patients with metastatic disease; (2) determine the extent to which varying levels of GCSF and GCSFR expression enhance CRC metastasis in a cell-intrinsic manner; and (3) determine the extent to which CRC GCSF signaling, systemic neutrophils, and tumor associated neutrophils interact to augment CRC metastasis. Under Aim 1, two possible correlations will be assessed: (1) between disease specific outcomes and GCSF levels in serum, and (2) between disease specific outcomes, and GCSF and GCSFR levels within the tumor microenvironment. Lentiviral transduction, shRNA, and CRISPR technology will be used in Aim 2 to modify human and murine CRC cells to over-express, downregulate, or abrogate both the receptor and ligand. This will allow quantitative evaluation of the influence of expression level on cell-intrinsic metastatic potential. Finally, in Aim 3, mouse models of micro- and macro-metastatic CRC will be used to determine how CRC GCSF signaling affects neutrophils (both systemic and tumor microenvironment) and how these interactions affect metastatic potential in vivo. This will extend the study beyond the clinical correlations and in vitro work to provide insight into the role of CRC GCSF signaling in the complex environment of an intact immune system. The approach is innovative, in the applicant's opinion, because it investigates a previously unexamined link between a pro-inflammatory cytokine that is highly over-expressed in human CRC and the role of tumor associated neutrophils in CRC using a combination of data from patients, as well as mechanistic in vitro and in vivo work. The approach is significant because it is expected to advance the study of CRC by producing a detailed understanding of the effects of a newly observed variation in human CRC. Specifically, the cell-intrinsic and cell-extrinsic effects of variable GCSF and GCSFR expression within CRC metastasis will be determined, advancing consideration of GCSF signaling as a therapeutic target in advanced CRC.